The Nrf2 Gene and Polypeptide
Nuclear factor erythroid-2 related factor 2 (NFE2L2; Nrf2), a cap-and-collar basic leucine zipper transcription factor, positively regulates a transcriptional program that maintains cellular redox homeostasis and protects cells from oxidative insult, including insult from chemotherapeutic agents (Rangasamy, et al. 2004. J Clin Invest 114, 1248). Nrf2 activates transcription of its target genes through binding specifically to the antioxidant-response element (ARE) found in those genes' promoters. The Nrf2-regulated transcriptional program includes a broad spectrum of genes, including antioxidants such as heme oxygenase-1, superoxide dismutase, glutathione reductase (GSR), glutathione peroxidase, thioredoxin, thioredoxin reductase, and peroxiredoxins (PRDX).
Lung Cancer
Lung cancer usually develops in the cells lining the lung's air passages. The two main types are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), based on the cell morphology. In non-small cell lung cancer, results of standard treatment are poor except for the most localized cancers. Surgery is the most potentially curative therapeutic option for this disease; radiation therapy can produce a cure in only a small number of patients and can provide palliation in most patients. Adjuvant chemotherapy may provide an additional benefit to patients with resected NSCLC. In advanced-stage disease, chemotherapy offers modest improvements in median survival, though overall survival is poor. Chemotherapy has produced short-term improvement in disease-related symptoms. Other forms of lung cancer include metastases of a primary cancer.
PCT Patent Publication No. WO 2006/128041 discloses siRNA molecules for Nrf2 and their use in treating cancer, preferably lung and kidney cancers. US Patent Application Publication No. 20020164576 discloses a method of inhibiting tumor growth (preferably a lymphoma cancer) using antisense molecules directed to Nrf2 or specific antibodies. Co-assigned US Patent Application Publication No. 20070042418, discloses the use of Nrf2 inhibitors including siRNA molecules for treating cancer. Co-assigned PCT Patent Publication No. WO 2008/114262 discloses certain Nrf2 siRNA molecules.
siRNA and RNA Interference
RNA interference (RNAi) is a phenomenon involving double-stranded (ds) RNA-dependent gene specific posttranscriptional silencing. Originally, attempts to study this phenomenon and to manipulate mammalian cells experimentally were frustrated by an active, non-specific antiviral defense mechanism which was activated in response to long dsRNA molecules (Gil et al. Apoptosis, 2000. 5:107-114). Later it was discovered that synthetic duplexes of 21 nucleotide RNAs could mediate gene specific RNAi in mammalian cells, without the stimulation of the generic antiviral defense mechanisms (see Elbashir et al. Nature 2001, 411:494-498 and Caplen et al. PNAS USA 2001, 98:9742-9747). As a result, small interfering RNAs (siRNAs), which are short double-stranded RNAs, have become powerful tools in attempting to understand gene function. Thus RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing in mammals mediated by small interfering RNAs (siRNAs) (Fire et al, Nature 1998. 391, 806) or microRNAs (miRNA; Ambros, Nature 2004 431:7006, 350-55; and Bartel, Cell. 2004. 116(2):281-97). The corresponding process in plants is commonly referred to as specific posttranscriptional gene silencing or RNA silencing and is referred to as quelling in fungi.
A siRNA is a double-stranded RNA molecule which inhibits, either partially or fully, the expression of a gene/mRNA of its endogenous or cellular counterpart, or of an exogenous gene such as a viral nucleic acid. The mechanism of RNA interference is detailed infra.
Studies have revealed that siRNA is effective in vivo in mammals, including humans. Bitko et al., showed that specific siRNAs directed against the respiratory syncytial virus (RSV) nucleocapsid N gene are effective in treating mice when administered intranasally (Nat. Med. 2005, 11(1):50-55). For reviews of therapeutic applications of siRNAs see for example Barik (Mol. Med 2005, 83: 764-773) and Chakraborty (Current Drug Targets 2007 8(3):469-82). In addition, clinical studies with short siRNAs that target the VEGFR1 receptor in order to treat age-related macular degeneration (AMD) have been conducted in human patients (Kaiser, Am J Ophthalmol. 2006 142(4)660-8). Further information on the use of siRNA as therapeutic agents is available, see for example Durcan (Mol. Pharma. 2008. 5(4):559-566), Kim and Rossi (BioTechniques 2008. 44:613-616) and Grimm and Kay (JCI, 2007. 117(12):3633-41).
Chemically Modified siRNA
The selection and synthesis of siRNA corresponding to known genes has been widely reported (see for example Ui-Tei et al., 2006. J Biomed Biotechnol. 2006: 65052; Chalk et al., 2004. BBRC. 319(1): 264-74; Sioud & Leirdal, 2004. Met. Mol Biol. 252:457-69; Levenkova et al., 2004, Bioinform. 20(3):430-2; Ui-Tei et al., 2004. NAR 32(3):936-48).
Examples for the use of, and production of, chemically modified siRNA are found in Braasch et al., 2003. Biochem., 42(26):7967-75; Chiu et al., 2003, RNA, 9(9):1034-48; PCT Patent Publications Nos. WO 2004/015107 and WO 02/44321. U.S. Pat. Nos. 5,898,031 and 6,107,094 teach chemically modified oligomers. U.S. Pat. No. 7,452,987 relates to compounds having alternating unmodified and 2′ sugar modified ribonucleotides. PCT Patent Application Nos. PCT/IL2008/000248 and PCT/IL2008/001197, assigned to the assignee of the present invention, and hereby incorporated by reference in their entirety, disclose chemically modified siRNA compounds.
Despite the evident progress, there remains a continued need for improved therapeutic molecules, in particular improved Nrf2 siRNA compounds, useful in treating cancerous or proliferative diseases, particularly lung cancers.